Engagement of multicomponent immunoreceptors such as the T cell antigen receptor (TCR) results in rapid activation of multiple protein tyrosine kinases (PTKs) including Lck, Fyn, ZAP-70 and Itk. These PTKs then phosphorylate a number of enzymes and adapter molecules involved in a complex signaling cascade. Our studies have focused on a critical substrate of the PTKs, LAT (linker for activation of T cells), a 36-38kD integral membrane protein. LAT is a critical transmembrane adaptor protein. We have performed studies to characterize how LAT is phosphorylated and binds a number of critical signaling molecules, thus bringing these other adaptor molecules and enzymes to the plasma membrane in the vicinity of the activated TCR. Biochemical, biophysical, genetic and microscopic techniques are currently employed to study the characteristics of LAT-based signaling complexes. A critical pathway activated after TCR engagement and primarily dependent on the LAT molecule is the ERK enzymatic pathway. A number of our older studies demonstrated how interaction of the enzyme phospholipase C gamma (PLCgamma) with a particular phosphorylated tyrosine of LAT results in activation of an enzyme cascade leading to ERK activation. In this pathway PLCgamma activation leads to breakdown of phosphoinositide lipids leading to diacylglycerol production. This product has a number of targets, one which is the enzyme RasGRP, which activates the small G protein Ras and thereby controls a cascade leading to ERK activation. This route to ERK activation is not the only pathway coupled to LAT leading to ERK activation. The adapter molecule Grb2 also binds phosphorylated LAT and brings the Ras activator molecule SOS to LAT as well. In many cell types SOS is the central Ras activator. The significance of SOS in T cells remains to be determined. To address SOS function this year we have successfully generated a mouse in which SOS can be deleted in T cells. We have begun a series of experiments designed to test the role of SOS in thymic development and peripheral T cell function. The previously generated SOS knock-out animals have proved to be not very useful since they demonstrate embryonic lethality. The mice we have made will prove to be an important reagent for many scientists since they allow for targeted SOS deletion in any cell or tissue of interest. In addition to biochemical and genetic studies of signaling molecules the laboratory has developed new methods of visualizing T cell activation using confocal microscopy. Many of the signaling molecules involved in the early TCR-coupled activation process have been tagged with fluorescent markers and expressed in T cells. The group has used these methods to observe the process of the assembly of signaling molecules into signaling clusters at the site of T cell activation. In studies underway this year we have studied the fate of LAT molecules. We have demonstrated that LAT is ubiquitinylated following T cell activation. Inhibition of this post-translational change results in prolonged LAT half-life and enhanced T cell signaling. These findings are under investigation in hopes of understanding how LAT ubiquitinylation may regulate T cell responses.